In vitro model of neurotrauma using the chick embryo to test regenerative bioimplantation.

Effective repair of spinal cord injury sites remains a major clinical challenge. One promising strategy is the implantation of multifunctional bioscaffolds to enhance nerve fibre growth, guide regenerating tissue and modulate scarring/inflammation processes. Given their multifunctional nature, such implants require testing in models which replicate the complex neuropathological responses of spinal injury sites. This is often achieved using live, adult animal models of spinal injury. However, these have substantial drawbacks for developmental testing, including the requirement for large numbers of animals, costly infrastructure, high levels of expertise and complex ethical processes. As an alternative, we show that organotypic spinal cord slices can be derived from the E14 chick embryo and cultured with high viability for at least 24 days, with major neural cell types detected. A transecting injury could be reproducibly introduced into the slices and characteristic neuropathological responses similar to those in adult spinal cord injury observed at the lesion margin. This included aligned astrocyte morphologies and upregulation of glial fibrillary acidic protein in astrocytes, microglial infiltration into the injury cavity and limited nerve fibre outgrowth. Bioimplantation of a clinical grade scaffold biomaterial was able to modulate these responses, disrupting the astrocyte barrier, enhancing nerve fibre growth and supporting immune cell invasion. Chick embryos are inexpensive and simple, requiring facile methods to generate the neurotrauma model. Our data show the chick embryo spinal cord slice system could be a replacement spinal injury model for laboratories developing new tissue engineering solutions.

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury.

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.

Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury.

Axon regeneration failure causes neurological deficits and long-term disability after spinal cord injury (SCI). Here, we found that the α2δ2 subunit of voltage-gated calcium channels negatively regulates axon growth and regeneration of corticospinal neurons, the cells that originate the corticospinal tract. Increased α2δ2 expression in corticospinal neurons contributed to loss of corticospinal regrowth ability during postnatal development and after SCI. In contrast, α2δ2 pharmacological blockade through gabapentin administration promoted corticospinal structural plasticity and regeneration in adulthood. Using an optogenetic strategy combined with in vivo electrophysiological recording, we demonstrated that regenerating corticospinal axons functionally integrate into spinal circuits. Mice administered gabapentin recovered upper extremity function after cervical SCI. Importantly, such recovery relies on reorganization of the corticospinal pathway, as chemogenetic silencing of injured corticospinal neurons transiently abrogated recovery. Thus, targeting α2δ2 with a clinically relevant treatment strategy aids repair of motor circuits after SCI.

Spinal SNAP-25 regulates membrane trafficking of GluA1-containing AMPA receptors in spinal injury-induced neuropathic pain in rats.

INTRODUCTION: Synaptosomal associated proteins of 25 kDa (SNAP-25), as a member of stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, is critical for membrane fusion and required for the release of neurotransmitters. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is implicated in pathologic pain. This study aimed to investigate whether and how SNAP-25 regulated AMPA receptors in neuropathic pain. METHODS: Male Sprague-Dawley rats underwent L4 spinal nerve ligation (SNL) or the sham procedure. After assessing mechanical allodynia and thermal sensitivity, the ipsilateral portion of the L4-5 spinal cord was harvested. The expression level of SNAP-25 was analyzed by Western blot analysis and real-time quantitative polymerase chain reaction. SNAP-25 phosphorylation and AMPA receptor membrane trafficking levels were evaluated with Western blot analysis. An association between SNAP-25 and AMPA membrane trafficking was confirmed by SNAP-25 expression or phosphorylation inhibition. RESULTS: The SNL procedure induced and maintained mechanical allodynia and thermal hyperalgesia. SNL increased the expression and phosphorylation of SNAP-25 and the membrane trafficking of AMPA receptors in the spinal cord. SNAP-25 expression or phosphorylation inhibition alleviated neuropathic pain and downregulated membrane trafficking of AMPA receptors after SNL. GluA1-containing AMPA receptor inhibition relieved mechanical allodynia and thermal hyperalgesia after SNL. CONCLUSIONS: The upregulation of SNAP-25-dependent membrane trafficking of AMPA receptors via SNAP-25 phosphorylation at Ser187 contributed to SNL-induced neuropathic pain. Thus, the inhibition of SNAP-25 expression or phosphorylation might serve as a treatment for neuropathic pain. However, the mechanism of GluA1-containing AMPA receptor membrane trafficking mediated by SNAP-25 phosphorylation in neuropathic pain deserves further exploration.

Changes in Nerve Growth Factor Expression and Macrophage Phenotype Following Intervertebral Disc Injury in Mice.

Nerve growth factor (NGF) is increased in intervertebral discs (IVDs) after disc injury and anti-NGF therapy improves low back pain in humans. Furthermore, M1 and M2 macrophage subtypes play a role in degenerative IVD injury. We examined M1 and M2 macrophage markers and NGF and cytokine expression in IVD-derived cells from control and IVD-injured mice for 28 days following injury. Ngf messenger RNA (mRNA) expression was increased 1 day after injury in injured compared with control mice, and persisted for up to 28 days. Flow cytometric analysis demonstrated that the proportion of F4/80+ CD11b+ cells was significantly increased from 1 day after injury for up to 28 days in injured compared to control mice. mRNA expression of M1 macrophage markers Tnfa, Il1b, and Nos2 was significantly increased 1 day after injury in injured compared to control mice, before gradually decreasing. At 28 days, no significant difference was observed in M1 markers. The M2a marker, Ym1, was significantly increased 1 day after injury in injured compared with control mice, while M2a and M2c markers Tgfb and Cd206 were significantly increased 7, 14, and 28 days after injury. Tumor necrosis factor α (TNF-α) and transforming growth factor ß (TGF-ß) stimulated Ngf mRNA and NGF protein expression in IVD cells. Our results suggest that TNF-α and TGF-ß may stimulate NGF production under inflammatory and non-inflammatory conditions following IVD injury. As TNF-α and TGF-ß are produced by M1 and M2 macrophages, further investigations are needed to reveal the role of macrophages in NGF expression following IVD injury. Our results may aid in developing treatments for IVD-related LBP pathology. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1798-1804, 2019.

Annulus fibrosus cell phenotypes in homeostasis and injury: implications for regenerative strategies.

Despite considerable efforts to develop cellular, molecular, and structural repair strategies and restore intervertebral disk function after injury, the basic biology underlying intervertebral disk healing remains poorly understood. Remarkably, little is known about the origins of cell populations residing within the annulus fibrosus, or their phenotypes, heterogeneity, and roles during healing. This review focuses on recent literature highlighting the intrinsic and extrinsic cell types of the annulus fibrosus in the context of the injury and healing environment. Spatial, morphological, functional, and transcriptional signatures of annulus fibrosus cells are reviewed, including inner and outer annulus fibrosus cells, which we propose to be referred to as annulocytes. The annulus also contains peripheral cells, interlamellar cells, and potential resident stem/progenitor cells, as well as macrophages, T lymphocytes, and mast cells following injury. Phases of annulus fibrosus healing include inflammation and recruitment of immune cells, cell proliferation, granulation tissue formation, and matrix remodeling. However, annulus fibrosus healing commonly involves limited remodeling, with granulation tissues remaining, and the development of chronic inflammatory states. Identifying annulus fibrosus cell phenotypes during health, injury, and degeneration will inform reparative regeneration strategies aimed at improving annulus fibrosus healing.

Ultrashortwave radiation promotes the recovery of spinal cord injury by inhibiting inflammation via suppression of the MK2/TNF­α pathway.

Mitogen­activated protein kinase­activated protein kinase 2 (MK2) and its mediated inflammation are involved in various diseases, including spinal cord injury (SCI). Ultrashortwave (USW) radiation has previously been reported to exert a protective effect on SCI. In the present study, through a series of reverse transcription­quantitative polymerase chain reaction (RT­qPCR), western blot and immunofluorescence assay, it was found that MK2 and tumor necrosis factor (TNF)­α/interleukin (IL)­1ß were elevated in patients with SCI and in H2O2­treated C8­D1A cells. Through gene level and protein level detection by using of RT­qPCR, western blot, immunofluorescence assay and terminal deoxynucleotidyl transferase (TdT) dUTP nick­end labeling assay, it was demonstrated that USW radiation inhibited the expression of MK2/TNF­α/IL­1ß and suppressed the apoptosis of H2O2­treated C8­D1A cells. Furthermore, it was confirmed that the overexpression of MK2 reversed the protective effect of USW on C8­D1A cells, which indicated that USW achieved its function via regulation of the MK2/TNF­α/IL­1ß pathway. Finally, using a constructed in vivo model and a series of RT­qPCR, western blot and IHC detection, it was confirmed that USW suppressed the expression of MK2 to promote functional recovery following SCI.

KLF6 and STAT3 co-occupy regulatory DNA and functionally synergize to promote axon growth in CNS neurons.

The failure of axon regeneration in the CNS limits recovery from damage and disease. Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration, but the underlying mechanisms are unclear. Here we show that forced expression of KLF6 promotes axon regeneration by corticospinal tract neurons in the injured spinal cord. RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in vitro, including sub-networks that were highly enriched for functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis, and bioenergetics. In addition, promoter analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting using ATAC-Seq data confirmed frequent co-occupancy. Co-expression of the two factors yielded a synergistic elevation of neurite growth in vitro. These data clarify the transcriptional control of axon growth and point the way toward novel interventions to promote CNS regeneration.

Spinal Astrocytic Thrombospondin-4 Induced by Excitatory Neuronal Signaling Mediates Pain After Facet Capsule Injury.

Thrombospondin-4 (TSP4) is a synaptogenic molecule that is upregulated in the spinal cord after painful facet joint injury and may contribute to spinal hyperexcitability. However, the mechanisms leading to increased spinal TSP4 are unclear. Because primary afferent activity is critical in the development of spinal hyperexcitability after facet joint injury, this study evaluated the role of afferent firing in the increase of spinal TSP4 and excitatory synapses. Intra-articular bupivacaine was administered immediately or 4 days after painful facet joint injury in male Holtzman rats, and TSP4 and excitatory synapses were quantified in the spinal cord at day 7. Immediate, but not delayed bupivacaine treatment, prevents the injury-induced increase in TSP4 and excitatory synapses in the dorsal horn (p < 0.0001). Preliminary in vitro experiments suggest that the excitatory signaling molecules ATP and glutamate may stimulate astrocytic TSP4 expression (p ≤ 0.04). Collectively, these results suggest that afferent activity early after facet joint injury is critical for the induction of spinal TSP4. This study advances the understanding of the timing and role of afferent activity in TSP4 expression after injury, which is critical for the therapeutic targeting of TSP4 to treat persistent pain conditions.

Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints.

OBJECTIVE: High-frequency, low-amplitude whole-body vibration (WBV) is being used to treat a range of musculoskeletal disorders; however, there is surprisingly limited knowledge regarding its effect(s) on joint tissues. This study was undertaken to examine the effects of repeated exposure to WBV on bone and joint tissues in an in vivo mouse model. METHODS: Ten-week-old male mice were exposed to vertical sinusoidal vibration under conditions that mimic those used clinically in humans (30 minutes per day, 5 days per week, at 45 Hz with peak acceleration at 0.3g). Following WBV, skeletal tissues were examined by micro-computed tomography, histologic analysis, and immunohistochemistry, and gene expression was quantified using real-time polymerase chain reaction. RESULTS: Following 4 weeks of WBV, intervertebral discs showed histologic hallmarks of degeneration in the annulus fibrosus, disruption of collagen organization, and increased cell death. Greater Mmp3 expression in the intervertebral disc, accompanied by enhanced collagen and aggrecan degradation, was found in mice exposed to WBV as compared to controls. Examination of the knee joints after 4 weeks of WBV revealed meniscal tears and focal damage to the articular cartilage, changes resembling osteoarthritis. Moreover, mice exposed to WBV also demonstrated greater Mmp13 gene expression and enhanced matrix metalloproteinase-mediated collagen and aggrecan degradation in articular cartilage as compared to controls. No changes in trabecular bone microarchitecture or density were detected in the proximal tibia. CONCLUSION: Our experiments reveal significant negative effects of WBV on joint tissues in a mouse model. These findings suggest the need for future studies of the effects of WBV on joint health in humans.

The roads to mitochondrial dysfunction in a rat model of posttraumatic syringomyelia.

The pathophysiology of posttraumatic syringomyelia is incompletely understood. We examined whether local ischemia occurs after spinal cord injury. If so, whether it causes neuronal mitochondrial dysfunction and depletion, and subsequent energy metabolism impairment results in cell starvation of energy and even cell death, contributing to the enlargement of the cavity. Local blood flow was measured in a rat model of posttraumatic syringomyelia that had received injections of quisqualic acid and kaolin. We found an 86 ± 11% reduction of local blood flow at C8 where a cyst formed at 6 weeks after syrinx induction procedure (P < 0.05), and no difference in blood flow rate between the laminectomy and intact controls. Electron microscopy confirmed irreversible neuronal mitochondrion depletion surrounding the cyst, but recoverable mitochondrial loses in laminectomy rats. Profound energy loss quantified in the spinal cord of syrinx animals, and less ATP and ADP decline observed in laminectomy rats. Our findings demonstrate that an excitotoxic injury induces local ischemia in the spinal cord and results in neuronal mitochondrial depletion, and profound ATP loss, contributing to syrinx enlargement. Ischemia did not occur following laminectomy induced trauma in which mitochondrial loss and decline in ATP were reversible. This confirms excitotoxic injury contributing to the pathology of posttraumatic syringomyelia.

SIRT1 Plays a Protective Role in Intervertebral Disc Degeneration in a Puncture-induced Rodent Model.

STUDY DESIGN: Experimental animal study of treatment of intervertebral disc (IVD) degeneration. OBJECTIVE: This report aims to evaluate the in vivo effects of SIRT1 on IVD biology and to explore its potential mechanism. SUMMARY OF BACKGROUND DATA: Silent mating type information regulator 2 homolog 1 (SIRT1) has attracted immense attention because of its functions in a variety of aging-related diseases. Despite previous studies indicated that SIRT1 showed a unique expression with degeneration in some in vitro study, there is no in vivo research on the role SIRT1 plays in IVD and its mechanism. METHODS: Coccygeal discs were punctured to induce disc degeneration. Sixteen C57BL/6J mice received either Carboxy methocel (Vehicle) or Resveratrol (RES) gavage. Eight SIRT1 mice and their SIRT1 littermates were also used in this study. At 2 and 6 weeks after puncture, magnetic resonance images were obtained. The mice were subsequently killed, and the spine was extracted for further evaluation. RESULTS: Coccygeal disc puncture caused IVD degeneration in the mice. A SIRT1 activator, RES, markedly ameliorated this pathological change, as demonstrated by stronger signal intensity in the T2-weighted images, as well as a significantly lower magnetic resonance imaging grade (at 2 wk vs. Vehicle group P < 0.001). Histological analysis also revealed an improvement in the RES group compared with the Vehicle group (P < 0.05). Genetic ablation of 1 allele significantly enhanced the level of damage relative to the wild-type mice. In addition, SIRT1 activation suppressed the expression of p16 and at the same time, promoted proliferating cell nuclear antigen and type II collagen expression in disc cells, whereas genetic ablation of 1 allele SIRT1 exhibited the opposite consequence. CONCLUSION: The SIRT1 activator RES protects against puncture-induced disc injury whereas SIRT1 deficiency aggravates tissue injury; the protective role of SIRT1 is partly mediated by suppressing p16, which plays a role in elevating the decreased proliferative ability of the senescent nucleus pulposus cells. LEVEL OF EVIDENCE: N/A.

Biocompatibility of a coacervate-based controlled release system for protein delivery to the injured spinal cord.

The efficacy of protein-based therapies for treating injured nervous tissue is limited by the short half-life of free proteins in the body. Affinity-based biomaterial delivery systems provide sustained release of proteins, thereby extending the efficacy of such therapies. Here, we investigated the biocompatibility of a novel coacervate delivery system based on poly(ethylene argininylaspartate diglyceride) (PEAD) and heparin in the damaged spinal cord. We found that the presence of the [PEAD:heparin] coacervate did not affect the macrophage response, glial scarring or nervous tissue loss, which are hallmarks of spinal cord injury. Moreover, the density of axons, including serotonergic axons, at the injury site and the recovery of motor and sensorimotor function were comparable in rats with and without the coacervate. These results revealed the biocompatibility of our delivery system and supported its potential to deliver therapeutic proteins to the injured nervous system.

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells.

BACKGROUND: Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury. RESULTS: Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H2O2 toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS. CONCLUSIONS: Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.

The dual role of astrocyte activation and reactive gliosis.

Astrocyte activation and reactive gliosis accompany most of the pathologies in the brain, spinal cord, and retina. Reactive gliosis has been described as constitutive, graded, multi-stage, and evolutionary conserved defensive astroglial reaction [Verkhratsky and Butt (2013) In: Glial Physiology and Pathophysiology]. A well- known feature of astrocyte activation and reactive gliosis are the increased production of intermediate filament proteins (also known as nanofilament proteins) and remodeling of the intermediate filament system of astrocytes. Activation of astrocytes is associated with changes in the expression of many genes and characteristic morphological hallmarks, and has important functional consequences in situations such as stroke, trauma, epilepsy, Alzheimer's disease (AD), and other neurodegenerative diseases. The impact of astrocyte activation and reactive gliosis on the pathogenesis of different neurological disorders is not yet fully understood but the available experimental evidence points to many beneficial aspects of astrocyte activation and reactive gliosis that range from isolation and sequestration of the affected region of the central nervous system (CNS) from the neighboring tissue that limits the lesion size to active neuroprotection and regulation of the CNS homeostasis in times of acute ischemic, osmotic, or other kinds of stress. The available experimental data from selected CNS pathologies suggest that if not resolved in time, reactive gliosis can exert inhibitory effects on several aspects of neuroplasticity and CNS regeneration and thus might become a target for future therapeutic interventions.

Nucleotide signaling in astrogliosis.

Acute and chronic damage to the central nervous system (CNS) releases large quantities of ATP. Whereas the ATP concentration in the extracellular space is normally in the micromolar range, under these conditions it increases to millimolar levels. A number of ligand-gated cationic channels termed P2X receptors (7 mammalian subtypes), and G protein-coupled P2Y receptors (8 mammalian subtypes) are located at astrocytes, as confirmed by the measurement of the respective mRNA and protein. Activation of both the P2X7 and P2Y1,2 subtypes identified at astrocytes initiates astrogliosis isolating damaged brain areas from surrounding healthy cells and synthesizing neurotrophins and pleotrophins that participate in neuronal recovery. Astrocytes are considered as cells of high plasticity which may alter their properties in a culture medium. Therefore, recent work concentrates on investigating nucleotide effects at in situ (acute brain slices) and in vivo astrocytes. A wealth of data relates to the involvement of purinergic mechanisms in astrogliosis induced by acute CNS injury such as mechanical trauma and hypoxia/ischemia. The released ATP may act within minutes as an excitotoxic molecule; at a longer time-scale within days it causes neuroinflammation. These effects sum up as necrosis/apoptosis on the one hand and proliferation on the other. Although the role of nucleotides in chronic neurodegenerative illnesses is not quite clear, it appears that they aggravate the consequences of the primary disease. Epilepsy and neuropathic pain are also associated with the release of ATP and a pathologic glia-neuron interaction leading to astrogliosis and cell death. In view of these considerations, P2 receptor antagonists may open new therapeutic vistas in all forms of acute and chronic CNS damage.

Signaling proteins in spinal parenchyma and dorsal root ganglion in rat with spinal injury-induced spasticity.

Development of progressive muscle spasticity resulting from spinal traumatic injury can be mediated by loss of local segmental inhibition and/or by an increased sensory afferent drive with resulting exacerbated α-motoneuron activity. To identify potential contributions of neuroactive substances in the development of such spasticity state, we employed a well-defined spinal injury-evoked spasticity rat model. Signaling molecules were analyzed in the spinal parenchyma below the level of spinal injury and in the corresponding dorsal root ganglion cells using Kinex™ antibody microarrays. The results uncovered the involvement of angiogenesis and neurodegeneration pathways together with direct cross-talk mediated by several hub proteins with SH-2 domains. At 2 and 5weeks after transection, up-regulation of several proteins including CaMKIV, RONα and PKCδ as well as MAPK3/ERK1 phosphorylation was observed in the spinal ventral horns. Our results indicate that these signaling molecules and their neuronal effector systems cannot only play an important role in the initiation but also in the maintenance of spasticity states after spinal trauma. The exclusivity of specific protein changes observed in lumbar spinal parenchyma but not in dorsal root ganglia indicates that new treatment strategies should primarily target specific spinal segments to prevent or attenuate spasticity states. BIOLOGICAL SIGNIFICANCE: Development of progressive muscle spasticity and rigidity represents a serious complication associated with spinal ischemic or traumatic injury. Signaling proteins, including their phosphorylation status, were analyzed in the spinal parenchyma below the level of spinal injury and in the corresponding dorsal root ganglion cells in a rat model of spinal injury using Kinex™ antibody microarrays. The results uncovered direct protein interaction mediated cross-talk between angiogenesis and neurodegeneration pathways, which may significantly contribute to the healing process in the damaged region. Importantly, we identified several target proteins exclusively observed in the spinal lumbar ventral horns, where such proteins may not only play an important role in the initiation but also in the maintenance of spasticity states after spinal trauma. Hence, potential new treatment strategies such as gene silencing or drug treatment should primarily target spinal parenchymal sites at and around the injury epicenter and most likely employ intrathecal or targeted spinal segment-specific vector or drug delivery. We believe that this work will stimulate future translational research, ultimately leading to the improvement of quality of life of patients with spinal traumatic injury.

Serotonin 2C receptor alternative splicing in a spinal cord injury model.

Spinal cord injury can have debilitating consequences, commonly resulting in motor dysfunction below the lesion site and the development of chronic pain syndromes. The serotonin pathway is important for inhibiting noxious stimuli and facilitating motor function after spinal cord injury. The serotonin 2C receptor (5HTR2C) has several characteristic features, and is regulated by the amount of serotonin 2C receptor as well as RNA editing and alternative splicing. In this study, we used a rat model of spinal contusion injury to investigate the relationship between the pain threshold and 5HTR2C alternative splicing. The pain threshold was assessed using mechanical stimulation with von Frey filaments. We then used real-time PCR to examine the RNA levels of 5HTR2C in three sections of the spinal cord: the rostral, injury-core, and caudal positions. On postoperative day 12, the pain threshold in injured rats was significantly reduced compared with sham-operated and naïve rats. The total 5HTR2C levels were significantly lower in injured rats than in naïve rats at all positions, and significantly lower in injured rats compared with sham-operated rats at injury-core and caudal positions. The ratio of exon Vb-skipped nonfunctional 5HTR2C mRNA to total 5HTR2C was significantly higher in injured rats compared with naïve rats at the injury-core and caudal positions, and significantly higher in injured rats compared with sham-operated rats at the caudal position. These results indicate that spinal contusion injury, which causes neuropathic pain, induces serotonergic dysfunction. This dysfunction appears to be mediated by decreased 5HTR2C mRNA expression, and alternative splicing. These results confirm the importance of considering splice variants when examining 5HTR2C.

Cloning and expression of a retinoic acid receptor ß2 subtype from the adult newt: evidence for an early role in tail and caudal spinal cord regeneration.

Retinoic acid receptor beta 2 (RARß2) has been proposed as an important receptor mediating retinoid-induced axonal growth and regeneration in developing mammalian spinal cord and brain. In urodele amphibians, organisms capable of extensive central nervous system (CNS) regeneration as adults, this receptor had not been isolated, nor had its function been characterized. We have cloned a full-length RARß2 cDNA from adult newt CNS. This receptor, NvRARß2, is expressed in various adult organs capable of regeneration, including the spinal cord. Interestingly, both the NvRARß2 mRNA and protein are up-regulated during the first 2 weeks after amputation of the tail, primarily in the ependymoglial and meningeal tissues near the rostral cut surface of the cord. Treatment with LE135, a RARß-selective antagonist, caused a significant inhibition of ependymal outgrowth and a decrease in tail regenerate length. These data support an early role for this receptor in caudal spinal cord and tail regeneration in this amphibian.